The object of this research is to increase our understanding of the mechanism of RNA polymerase. We shall use various spectroscopic techniques (circular dichroism (CD), absorption spectra, fluorescence, and fluorescence-detected circular dichroism (FDCD)) to study the conformation of RNA polymerase and its complexes with DNA templates and substrates. In addition to the intrinsic properties of the enzyme and template, various spectroscopic probes will be used. Some of these bind noncovalently but very tightly: rifampicin and a fluorescence-tagged rifamycin, Rose Bengal. Other probes will be introduced by chemical modification: fluorescein and eosin via their isothiocyantes or iodoacetamide derivatives, pyridoxal phosphate. The intrinsic and extrinsic probes will be used to follow the conformational state and kinetics of reaction as the enzyme binds to DNA and as nucleotides or dinucleotides bind to form initiation and elongation complexes. The subunit localization of these probes will be determined and used to obtain information about the role of various subunits. The involvement of specific groups in substrate binding and catalysis will be explored by photoaffinity labeling with 8-azido ATP. Most of the extrinsic probes which we propose to use can also serve as catalysts for photo-oxidation, which will be used to locate and identify functionally significant groups. In addition to the native E. coli enzyme, the altered or modified enzyme produced by ADP-ribosylation in T4-infected E. coli cellls will be studied to determine the effect of ADP-ribosylation on protein conformation and on the equilibrium and kinetics of template binding.